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TCSPC Fluorescence Lifetime Spectrofluorometer PicoMaster Photon Technology International
TCSPC Fluorescence Lifetime Spectrofluorometer
PicoMaster
Photon Technology International
PicoMaster
Applications of time-resolved fluorescence (TRF) have been growing very rapidly in recent years. They encompass very diverse disciplines, ranging from chemistry and biology to various materials science disciplines such as nanotechnology, semiconductors, crystals, glasses and ceramics. These applications are based on the fluorescence lifetime, which is the average time a molecule spends in the excited state before emitting a photon and returning to the ground state.
The fluorescence lifetime provides complementary information to the commonly used steady state measurements, as it adds the ability to resolve molecular dynamic events and multiple components of a system, which are averaged in the steady state experiment. The lifetime not only reflects intrinsic properties of the excited molecule, but is also affected strongly by properties of the environment and by various interactions with surrounding molecules.
For example, conformational changes in proteins, nucleic acids or other macromolecules can be monitored and detected by measuring the emission decay of the probe molecule. Distances between two chromophore groups of a macromolecule can be determined by studying the fluorescence resonance energy transfer (FRET), which reduces the lifetime of the donor molecule. Polarity, viscosity of the environment, ion transport, and local electric field will be “imprinted” in the lifetime of the excited molecule. The presence of impurity or a dopant will affect the luminescence decay of a crystal.
Emission decays often show more than one lifetime and in some cases are described by even more complex kinetic behavior. PTI systems include a complete software package, which contains analysis modules for virtually any time-resolved application. Another important technique is time-resolved emission anisotropy, which is an emission decay measurement with polarized light. The temporal behavior of anisotropy depends on rotational freedom of the emitting molecule and thus can be used to determine microviscosity of a membrane, the size of a protein, to follow folding and unfolding of a protein or to monitor curing processes of polymers.
Fluorescence decays shown in the adjacent figure illustrate a complex decay of intrinsic tryptophan (Trp) in bovine serum albumin (BSA) protein measured across the emission spectrum. The analysis reveals 3 lifetime components pointing to heterogeneous environment and conformational diversity of the protein imprinted on the fluorescence decay of polarity sensitive Trp. Such detailed information is not revealed in the steady state measurement alone.
Fluorescence Lifetime Spectrometer
PTI has been manufacturing modular fluorescence systems for nearly three decades. Fluorescence lifetime instrumentation has always been one of the main product lines of our company. In fact, PTI was awarded a prestigious R&D 100 Award for developing a new stroboscopic lifetime technique back in 1989. Our team of scientists and engineers understands that diverse applications require different experimental approaches and therefore we develop and offer different techniques that can meet these challenges.
The PicoMaster™ line represents modular fluorescence lifetime systems based on the Time Correlated Single Photon Counting (TCSPC) technique. These instruments can meet the strictest demands with regard to the lifetime range, from single picoseconds
to seconds, and provide the highest dynamic range of at least 5 orders of magnitude. There are many types of light sources that the PicoMaster™ can use, such as pulsed LEDs, laser diodes, and mode-locked lasers pumping OPO or dye lasers and higher harmonics generators. With its Multi-Channel Scaling (MCS) capability, even low repetition lasers, such as nitrogen/dye or Q-switched can be used for longer fluorescence or phosphorescence lifetimes. A variety of detectors are available that can cover different spectral ranges, from UV to NIR, making the instruments suitable for very diverse applications. And being modular, they can be combined with the steady state fluorometer, phosphorescence system or utilized in microscopy measurements.
Fluorescence Lifetime Measurements
Fluorescence decays of bovine serum albumin protein measured with PicoMaster™ 1 (295 nm LED and PMD-2 fast PMT detector). A 3-exponential fitting function required to obtain adequate fits (pre-exponential factors in parentheses):320 nm: 0.3ns (0.43), 2.6ns (0.23) and 6.5ns (0.34)340 nm: 0.3ns (0.20), 2.8ns (0.22) and 6.5ns (0.58)420 nm: 0.4ns (0.34), 4.6ns (0.38) and 7.9ns (0.28)
Protein decay, ex=295 nm, em=320, 340, 400 nm
Signal Statistics - Due to a low probability of photon detection in a single excitation event, the standard deviation of the measured intensity is governed by Poisson statistics. Therefore, the S/N is well-defined and statistical data analysis involving numerical reconvolution and fitting of various decay models can be performed in a rigorous and reliable way.
High Dynamic Range - Photons can be counted practically indefinitely, so fluorescence decays can be obtained and analyzed over many orders of magnitude (typically 4 to 5), which enhances the precision of the lifetime determination, especially important for complex decays.
Short Lifetimes - Due to availability of lasers with ultra-short pulses (fs-ps), low-jitter multi-channel plate (MCP) detectors and single photon avalanche photodiodes (SPAD), lifetimes of a few picoseconds can in principle be measured.
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Lifetimes in NIR - Due to availability of photon counting PMT and SPAD detectors with near-infrared sensitivity, the TCSPC is the best choice to measure pico and nanosecond lifetimes in NIR.
Time Domain Technique - Fluorescence intensity is measured directly as a function of time, which makes it more intuitive to interpret and easy to follow the kinetic mechanism of a system during the measurement.
Immune to Intensity Fluctuations - Since no more than a single photon is counted as a result of a single flash, the intensity vs. time histogram is not affected by pulse-to-pulse intensity fluctuations of the light source.
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The Advantages of TCSPC
PicoMaster
Modern TCSPC Technology
The TCSPC systems offered by PTI represent the most modern state of the art technology.
Gone are the days when the TCSPC electronics was mounted in a huge, floor standing NIM-BIN rack that housed a collection of stand-alone modules like the time-to-amplitude converter (TAC), two constant fraction discriminators (CFD), time calibrator, coaxial cable-based delay line, a web of interconnecting cables and an assortment of knobs and toggle-switches.
In the modern PTI offering, all these components, including the TAC, two CFDs, a fast analog-to-digital converter (ADC) and the memory buffer are all integrated in a single PCI board. The board also provides two 12 V outputs to power the PMT and an optional trigger photodiode. The only connections to the board are the START and
STOP pulses and an optional external TTL trigger. All the board functions are conveniently controlled from Felix GX software – a comprehensive software platform that operates all PTI instruments and data analysis.
The TCSPC board also has a Multi-Channel Scaling (MCS) function incorporated. That makes the TCSPC system really versatile and capable of measuring longer fluorescence and phosphorescence lifetimes, from a few tens of nanoseconds to seconds. With the MCS operation the user can utilize low repetition rate sources, such as xenon flash lamps as well as nitrogen and Q-switched lasers.
PTI TCSPC systems offer femtosecond time resolution, MHz count rates and are excellent choices for the most demanding applications.
Max Count Rate: 10 MHz
Repetition Rates: 0 to 200 MHz
Frequency Divider: 1-2-4
Dead Time: 100 ns
Min Time/Channel: 813 fs
Max Time Channels/Curve: 4096
Measurement Times Down to: 0.1 ms
TAC Range: 50 ns to 5 µs
Biased TAC Amplifier Gain: 1 to 15
CFD Threshold (Photon Channel): -20 to -500 mV
CFD Threshold (Sync Channel): -20 to -500 mV
Sync Delay: Electronic, automatically calculated and applied for the selected TAC time range
MCS min time/channel: 25 ns
Old TCSPC electronics
Modern TCSPC board
PicoMaster
TCSPC Technique
The TCSPC is the time-domain technique. Like the other PTI lifetime technique, the Strobe, it utilizes pulsed light sources (lasers, LEDs and old-fashioned ns flash lamps). It also measures the same experimental functions as the Strobe, i.e. the fluorescence decay and the IRF. Its detection, however, is based on a different principle.
The block diagram shows the principle of the TCSPC operation. The pulse generator triggers the light source and also outputs a sync START pulse which triggers the detection electronics. Alternatively, for the ultimate in temporal resolution, the START pulse can be generated by a fast photodiode in front of the excitation source.
The START pulse after being fed through a Constant Fraction Discriminator (CFD) starts the Time-to-Amplitude Converter (TAC), the key element of the technique. After being triggered, the TAC starts a voltage ramp, which is linear in time.
In the mean time, the sample has been excited and has emitted fluorescence photons. When the PMT detects the 1st photon, a short pulse is created at the output of the PMT. The photoelectron pulses from the PMT show considerable pulse-to-pulse amplitude variations and are therefore fed through another CFD, which eliminates the time jitter caused by the amplitude spread. The signal then enters the TAC as a STOP pulse and stops the voltage ramp.
The voltage value (equivalent to the time difference between the start and stop pulses) is read by an analog-to-digital converter (ADC), converted to a time channel and the count value in that time channel is incremented by 1. The cycle is repeated with each flash and eventually after many cycles a histogram of counts vs. time channels is created. If the photon detection rate is low enough, so no more than a single photon is detected per cycle, the histogram represents undistorted fluorescence decay.
An important feature is that the single photon counting obeys the Poisson statistics. Because of that, the standard deviation of each data point is well determined, i.e. σ = N1/2, where N is the number of counts. This makes the data precision very predictable and facilitates the analysis process, where the knowledge of standard deviations is required.
As in the Strobe technique, in most cases the analysis requires that the decay and the IRF are collected. The model parameters (e.g. lifetimes and pre-exponential factors) are recovered from the non-linear least squares fitting procedure that involves iterative re-convolution of the IRF and model function.
Excitation Sources
Nanosecond LED and LD Light Sources
PicoMaster
The PicoMaster™1 system operates with nanosecond pulsed light emitting diodes (LED) and nanosecond laser diodes (LD). LEDs and LDs are very stable, versatile, inexpensive and maintenance free light sources. They are available from UV to NIR and can cover most of
fluorescence lifetime applications. The PicoMaster™1 comes with an LED/LD pulser, which can operate any of the LEDs and LDs listed.
LED: 266, 280, 297, 310, 340, 368, 375, 403, 407, 432, 444, 456, 486, 510, 572 nm
LDs: 633, 649, 667 nm
Rep Rate: up to 180 kHz (LED/LD dependent)
Pulse Width: < 1.5 ns (LED/LD dependent)
LED and LD Spectra (Normalized)
PicoMaster
Excitation Sources
Picosecond LD Light Sources
The PicoMaster™2 utilizes picosecond laser diodes. A number of LD heads are available from with wavelengths ranging from UV to NIR. The pulse width can be as short as 50 ps, which is sufficient to
measure lifetimes down to about 10 ps. The ps LDs can operate at very high rep rates.
picosecond Laser Diodes:
375, 405, 445, 473, 488, 635, 650, 660, 670 nm (up to 1550 nm available)
LDs: 633, 649, 667nm
Rep Rate: Variable: 1, 20, 50 MHz (up to 80 MHz available)
Pulse Width: down to 50 ps
Picosecond/Femtosecond Lasers
Other optional pulsed light sources will include mode-locked argon ion, Nd: YAG or Ti: Sapphire lasers. Argon ion and Nd: YAG provide single wavelength outputs and usually need an additional dye laser and a frequency doubler. The Ti: sapphire laser is much more versatile and stable. It self-mode locks, runs at rep rates around 80 MHz and is tunable over ca. 700-1100 nm range. Its output can be frequency doubled or tripled with a SHG or THG crystal. In addition, optical
parametric oscillators (OPO) can be used to broaden the excitation range of these lasers. These lasers will be at their best when used with a fast MCP detector and when combined with the PicoMaster-2 will provide capability of measuring lifetimes of a few ps.
These ultra-fast lasers are available on special requests. Please contact a sales representative in your area for more details.
Nanosecond LED and LD Light Sources Relative Intensities
The fundamental requirement of the TCSPC measurement is that the photon count rate must be low enough in order to avoid multiple photon detection, typically not exceeding 3-5% of the excitation repetition rate. At higher count rates, the measured decay curve will become distorted.
This eliminates relatively inexpensive low rep light sources, such as nitrogen/dye, Q-switched and excimer lasers as viable excitation sources for the TCSPC. On the other hand, these sources are excellent choices for PTI’s Strobe Technique and are widely used with TM-3 LaserStrobe™system. The lowest practical rep rates for TCSPC light sources should start at about 10 kilohertz and higher.
The ultrafast light sources operating at rep rates of tens of MHz seem ideal for TCSPC, as the decay acquisition can be completed
in seconds or even faster if the sample intensity is reasonable. The TCSPC electronics in PTI systems has a dead time of only 100 ns and can count photons at up to 10 MHz rates, which is more than enough for the fastest lasers available.
With the high rep rate sources the range of lifetimes that can be measured becomes limited. For a laser operating at 80 MHz, the time period between flashes is only 12.5 ns, so only the lifetimes shorter than about 2 ns can be measured. For longer lifetimes the rep rate has to be reduced and the acquisition times will become longer. A pulse picker or a cavity dumper to reduce the rep rate is necessary for mode-locked ps/fs lasers to ensure that the useful range of lifetimes can be measured. The pulsed laser diodes and LEDs from PTI have rep rate control built in.
Pulse Pile-up Effect, Rep Rate and Speed
PicoMaster
The PicoMaster systems are excellent choices for measuring all possible ranges of fluorescence lifetimes. Depending on the selection of the excitation source and the detector, the TCSPC mode of operation will cover lifetimes from single picoseconds to microseconds.
For those users who work in the area of inorganic luminescence or phosphorescence where the lifetimes are much longer, the TCSPC board has a built-in Multi-Channel Scaling (MCS) capability, which
provides the lifetime range from tens of nanoseconds to seconds, depending on the rep rate of the excitation source. Switching from TCSPC to MCS operation is done in the software and the TTL trigger for the MCS is provided either from the light source pulser or from the ASOC-10 system interface. The MCS mode will work well with low rep rate sources such as PTI nitrogen/dye laser, Xe flash lamp as well as with LEDs and LDs operating at kHz frequencies.
Lifetime Range
Long fluorescence decay of Ru(bpy)3 measured with TL460 LED and R928 pmt in 920C cooled pmt housing.
Ruthenium Bipyridyl
Short lifetimes measured with TD375 ps laser diode and PMD-2 fast pmt. Samples: curcumin in cyclohexane (43 ps), erythrosine in H2O (89 ps), erythrosine in MeOH (465 ps) and POPOP in EtOH (1.29 ns).
Short Lifetimes
Luminescence decay of Tb+3 ion measured with Xe flash lamp excitation and R928 pmt in 920C cooled pmt housing using the Multi-Channel Scaling (MCS) mode.
Tb MCS
PicoMaster
Spectral Range
Software Control
Excitation
The PicoMaster™1 uses PTI proprietary nanosecond LED and LD sources, which cover the excitation range from 260 to 670 nm. There are 19 different LED/LD to choose from, which ensures that there is a proper excitation source for practically any sample.
The PicoMaster™2 uses picosecond LDs, which are available from 375 to 1550 nm. Although they lack the deep UV coverage that is
available with ns LEDs, the main benefit here is the short pulse, higher energy and higher rep rates.
Other light sources, like ultra-short mode-locked lasers with higher harmonics generators are available on request. These sources, depending on the choice, will combine broad excitation wavelength coverage with the ability to measure very short lifetimes.
Emission
The PicoMaster™ systems can accommodate a broad range of detectors. For UV-VIS there are two fast PMT detectors that cover the ranges of 185-650 nm and 185-820 nm, respectively. A number of side-on PMTs used with our QuantaMaster™ systems can also be used as TCSPC detectors, especially if ordered with the TE-cooled
920C housing. Depending on the choice, these PMTs will cover the range of 185-1200 nm.
There are four PMT detectors that will extend the detection range into NIR, either to 1400 nm or 1700 nm. Two of these PMTs will also cover UV-VIS range starting from 300 nm.
PicoMaster™ comes with FelixGX software for instrumental control and includes new PowerFit-10 analytical software for fluorescence lifetime analysis.
Through the new, powerful ASOC-10 USB interface FelixGX provides a full set of data acquisition protocols and controls the hardware for all system configurations and operating modes.
FelixGX controls:
Monochromators
Motorized slits
Motorized polarizers
Motorized sample holders
Temperature control Peltier devices
Detectors
External devices such as stopped flow or titrator
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FelixGX also fully controls all functions of the TCSPC board:
Time-to-Amplitude Converter (TAC):
Range
Gain
Offset
Limit low and limit high
Sync and Stop channel
Discriminator threshold
Zero crossing level
Time delay of Sync pulse
Automatically calculated or
User defined
Number of channels
Frequency divider 1, 2 or 4
Acquisition stop method (stop button, peak channel count or time)
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PicoMaster
Lifetime Analysis
PowerFit-10 software (incorporated into FelixGX) provides powerful analytical package for decay data analysis. All modules include reconvolution algorithms, selection of data weighing (Poisson statistics or analog signals), shift and offset parameters, statistical goodness-of-
fit parameters (Chi-square, Durbin-Watson, Runs test, residuals and autocorrelation), as well as standard deviations of the fit parameters.
BSA 420 nm decay
Protein decay analyzed with a multiexponential model function. At least 3 lifetimes are required to adequately fit the decay as evidenced by chi-square, weighed residuals and autocorrelation’
PowerFit-10 contains the following modules:
Multi-exponential (1 to 4 lifetimes) decay It fits decay to up to 4 lifetimes and corresponding pre-exponential factors, the lifetimes can be floating or individually fixed.
Multi-file multi-exponential 1 to 4 exponential fitting program operating in a batch mode, will analyze up to 100 fluorescence decays.
Global (1 to 4 lifetimes) analysis Analyzes simultaneously several decay data sets assuming that the lifetimes are the same for all data sets and only pre-exponential factors vary.
Anisotropy decay Free rotor, restricted rotor and other models.
Stretched exponential General fitting function which is applicable to a variety of complex kinetics, such as energy transfer in diffusion-controlled systems (e.g. Yokota –Tanimoto model), restricted geometries (molecules on surfaces, zeolites) etc.
Micelle quenching kinetics Applies to fluorophores in micelles in the presence of external quenchers. The decay kinetics follows Infelta-Groetzel-Tachiya model, which allows the determination of micelle aggregation number and diffusion-controlled quenching rate constant.
Exponential Series Method (ESM) Unconstrained lifetime distribution analysis which uses up to 200 exponential terms with logarithmically spaced lifetimes. Allows for negative pre-exponentials (risetimes).
Maximum Entropy Method (MEM) Unconstrained lifetime distribution analysis (up to 200 exponential terms with logarithmically spaced lifetimes) using Shannon-Jaynes entropy function – bias free approach to data analysis. Allows for negative pre-exponentials (risetimes).
Time-Resolved Spectra (TRES)
Decay-Associated Spectra (DAS)
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PicoMaster
Convolution and Deconvolution
Because the excitation pulse is not infinitely narrow in time, it is necessary to correct for the distorting effect of the instrument response function (IRF, which comprises the light source width and detector response) on the decay data. This process, called deconvolution, requires the light source profile and the fluorescence decay data. For fitting a decay model, e.g. one or more exponentials, the method of choice world-wide appears to be iterative reconvolution. The
light source profile is convoluted with a trial decay function and the parameters varied until the best fit is obtained to the actual data. PTI’s software accomplishes this rapidly and efficiently. All PTI decay data analysis programs include deconvolution at some stage of the analysis. If the decay lifetimes are much longer than the IRF width, the user has an option of skipping deconvolution.
Lifetime Distribution Analysis Programs
There is a growing interest in the recovery of distributions of fluorescence lifetimes from decay data. PTI offers two programs, which quickly accomplish this mathematically complex task. The Exponential Series Method (ESM) involves fitting the data to a large sum (up to 200) of exponentials with fixed, logarithmically spaced lifetimes and variable pre-exponential coefficients by minimizing Chi-square. The Maximum Entropy Method (MEM) uses the same trial function but maximizes the Shannon-Jaynes entropy subject to a constraint
on Chi-square. Both methods are model-free and start with a flat distribution function. The MEM is the most rigorous data analysis method and offers bias free solution to a problem of fitting complex multi-parameter decays. As experimentalists study systems of ever increasing complexity (proteins, membranes, micelles, molecules on surface, nanoparticles etc.), these distribution analysis programs will prove invaluable and in fact necessary.
Fluorescence decay of CdSe quantum dots in chloroform measured with 490 nm pulsed LED excitation and monitored at 580 nm. The decay analysis with MEM reveals a broad bimodal lifetime distribution reflecting polidispersity of QDots.
Fluorescence decay of CdSe quantum dots
PicoMaster
Specifications
System
PicoMaster 1 PicoMaster 2
Detection Technique
Time-Correlated Single Photon Counting (TCSPC) Time-Correlated Single Photon Counting (TCSPC)
Light Source Range
LEDs: 266, 280, 297, 310, 340, 368, 375, 403, 407, 432, 444, 456, 486, 510, 518, 572 nm
LDs: 633, 649, 667 nm
picosecond Laser Diodes: 375, 405, 445, 473, 488, 635, 650, 660, 670 nm
(up to 1550 nm available)
Rep Rate
up to 180 kHz (LED dependent) Variable, up to 100 MHz
Pulse Width
< 1.5 ns (LED dependent) Down to 50 ps
PMT Response
180 ps (UV-VIS), 300 ps (NIR) 180 ps (UV-VIS), 300 ps (NIR)
MCP Response
25 ps
Temporal Resolution
813 fs 813 fs
Shortest Lifetime
40 ps < 10 ps
Emission Spectral Range
180–1700 nm (detector dependent) 180–1700 nm (detector dependent)
PTI has a policy of continuous product development and reserve the right to amend specifications without prior notice (Dec 2010)
USA: Photon Technology International, Inc., 300 Birmingham Road, PO Box 272 Birmingham, NJ 08011 Tel: 609-894-4420, Fax: 609-894-1579, E-mail: [email protected], www.pti-nj.com
Canada: Photon Technology International, Inc., 347 Consortium Court, London, Ontario, N6E 2S8Tel: 519-668-6920, Fax: 519-668-8437, E-mail: [email protected]
UK: Photon Technology International, Inc., Unit M1 Rudford Industrial Estate, Ford Road, Ford, West Sussex BN180BFTel: +44 (0) 1903 719555, Fax: +44 (0) 1903 725722. E-mail: [email protected]
Germany: PhotoMed GmbH, Inninger Str. 1, 82229 Seefeld, Germany,Tel: +49 (0) 8152 993090, Fax: +49 (0) 8152 993098, E-mail: [email protected] Technology International
Complete Line of Fluorescence Spectroscopy Instruments from PTI
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Steady State Fluorescence and Phosphorescence Spectrofluorometers
TimeMaster™ Series
Fluorescence Lifetime Spectrofluorometers
RatioMaster™ Series
Fluorescence Microscopy Spectrofluorometers
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